The overall goal of this proposal is to examine whether protons can serve as co-transmitters in cholinergic neuronal synaptic signaling. The rationale for this possibility carne from our recent findings that acidification uniquely modulated the recombinant neuronal nicotinic acetylcholine receptors (nAChRs) in part based on their desensitization kinetics and their beta subunit composition. In sharp contrast to reports that acidification generally inhibits ligand- and voltage-gated cation channels, we found that rapid (approximately 20 ms) acidification enhanced the alpha3/beta4 neuronal nAChR current and accelerated its activation and decay kinetics. Since it is well known that: a) repetitive firing in the brain causes brief acidic shifts, b) ACh is stored in acidic vesicles, and c) release of vesicular protons inhibits presynaptic Ca2+ channels, we hypothesize that brief intermittent acidification of synaptic cleft modulates the postsynaptic nAChRs providing focal plasticity to synaptic signaling. The experiments proposed here are aimed at: 1) examining, on a time scale approximating synaptic transmission, how acidification modulates the native neuronal nAChRs in primary culture of chromaffin cells, cortical, and cerebellar neurons; 2) imaging the microdomains of acidification and probing for presence of "proton sparks" with transmitter release in PC12 and chromaffin cell cultures; 3) evaluating directly the roles of released protons in modulation of synaptic signaling in cortical and cerebellar neurons. To test this hypothesis under feasible experimental conditions, we will use two models of neurosecretion: 1) PC12 and chromaffin cells and 2) cultures of cortical and cerebellar neurons, where agonist and protons may be applied rapidly (approximately 2 ms) and briefly, and membrane currents measured simultaneously. In chromaffin cells and cortical neurons, we shall quantify the effect of protons in modulation of nAChRs, and image the acidification profiles resulting from the release of secretory vesicles into paracellular space, using custom-made pH-sensitive dyes, in combination with rapid (240-480 f/s) confocal microscopy and a novel high-resolution optical technique (Total Internal Reflection Fluorescence Microscopy, TIRF), developed in our laboratory. Our finding, we believe, will provide novel insights into synaptic signaling that may be critical in the pathogenesis of neurodegenerative diseases such as Alzheimer's, where proton content of synaptic vesicle may be altered leading to impaired cholinergic signaling.